Diabetic foot ulceration (DFU) is a major problem that significantly impairs quality of life of the patient, leads to prolonged hospitalizations and may require major amputation, demonstrating the urgent need to develop next generation treatments for these and other chronic wounds. To fill this critical gap in patient care, novel sources of autologous cells that are more repair-competent are immediately needed. Our long term goal is to develop a therapeutic approach, based on induced pluripotent stem cell (iPSC) technologies, that will reverse DFU fibroblasts from a non-healing to a healing phenotype by treating these cells in situ, directly in the patient's wound without needing to remove and culture them. To lay the groundwork for this, the immediate goal of our project is to greatly improve the wound repair potency of DFU fibroblasts following their reprogramming to iPSC, that will reveal how epigenetic memory (patterns of DNA methylation retained from the cell type reprogrammed) can best be exploited to acquire a spectrum of pleiotrophic effects that will trigger wound repair. In light of the emergence of epigenetics as a critical regulator of the metabolic memory linked to diabetic cell dysfunction, DFU fibroblasts are likely to be controlled by epigenetic mechanisms (miRNA, histone modification and DNA methylation). As a result, we expect iPSC reprogramming to reset the epigenome and reverse metabolic memory to improve repair after iPSC differentiation. The overall objective is to develop new sources of autologous, repair-competent fibroblasts using iPSC technologies that will dramatically improve DFU therapy. Our central hypothesis is that DFU fibroblasts will become repair- competent when reprogrammed to iPSC and subsequently differentiated to a fibroblast lineage by acquiring repair-promoting functions that will be mediated by epigenetic controls. The rationale for our research is based on exciting new preliminary data that has established the augmented healing potential of iPSC-derived fibroblasts (iPDK) and in vivo models of diabetic wound repair developed in our labs that will determine this repair potential. To test this hypothesis, we have developed skills and data that support the feasibility of this approach by establishing that iPSC-derived fibroblasts are more versatile than their parental fibroblasts. We plan to test our central hypothesis by pursuing the following specific AIMS: AIM 1-Identify cellular functions that trigger repair-competency when repair-deficient, DFU fibroblasts are reprogrammed to iPSC and differentiated to fibroblasts, AIM 2-Reveal if the switch to repair-competency is linked to distinct DNA methylation profiles, histone modifications or miRNA signatures that regulate repair functions in iPDK fibroblasts, and AIM 3- Establish repair efficacy of iPSC-derived fibroblasts in two, well-established animal models of diabetes and optimize their delivery to heal diabetic wounds. Ultimately, this knowledge has the potential to transform the care of patients suffering from DFUs and will be widely applicable to many other types of non- healing wounds, as well as to periodontal disease and aging-related diseases.